1. Technical Field
The present invention relates to a method for producing an L-amino acid by fermentation, and more specifically to a gene derived from Escherichia coli which aids in this fermentation. The gene is useful for improvement of L-amino acid production, and specifically, for example, for L-threonine production.
2. Description of the Related Art
Conventionally, L-amino acids are industrially produced by fermentation methods utilizing strains of microorganisms obtained from natural sources or mutants thereof, which are modified to enhance production yields of L-amino acids.
Many techniques to enhance production yields of L-amino acids have been reported, including transformation of microorganisms with recombinant DNA (see, for example, U.S. Pat. No. 4,278,765). Other techniques for enhancing production yields include increasing the activities of enzymes involved in amino acid biosynthesis and/or desensitizing the target enzymes of the feedback inhibition by the resulting L-amino acid (see, for example, WO 95/16042 or U.S. Pat. Nos. 4,346,170, 5,661,012 and 6,040,160).
Strains useful in production of L-threonine by fermentation are known, including strains with increased activities of enzymes involved in L-threonine biosynthesis (U.S. Pat. Nos. 5,175,107; 5,661,012; 5,705,371; 5,939,307; EP0219027), strains resistant to chemicals such as L-threonine and its analogs (WO0114525A1, EP301572A2, U.S. Pat. No. 5,376,538), strains with target enzymes desensitized to feedback inhibition by the produced L-amino acid or its by-products (U.S. Pat. Nos. 5,175,107; 5,661,012), and strains with inactivated threonine degradation enzymes (U.S. Pat. Nos. 5,939,307; 6,297,031).
The known threonine-producing strain VKPM B-3996 (U.S. Pat. Nos. 5,175,107, and 5,705,371) is the best threonine producer known at present. For construction of the strain VKPM B-3996, several mutations and a plasmid, described below, were introduced into parent strain E. coli K-12 (VKPM B-7). Mutant thrA gene (mutation thrA442) encodes aspartokinase homoserine dehydrogenase I, which is resistant to feedback inhibition by threonine. Mutant ilvA gene (mutation ilvA442) encodes threonine deaminase having decreased activity which results in a decreased rate of isoleucine biosynthesis and to a leaky phenotype of isoleucine starvation. In bacteria containing the ilvA442 mutation, transcription of the thrABC operon is not repressed by isoleucine, and therefore is very efficient for threonine production. Inactivation of the tdh gene results in prevention of threonine degradation. The genetic determinant of saccharose assimilation (scrKYABR genes) was transferred to said strain. To increase expression of the genes controlling threonine biosynthesis, plasmid pVIC40 containing mutant threonine operon thrA442BC was introduced in the intermediate strain TDH6. The amount of L-threonine accumulated during fermentation of the strain can be up to 85 g/l.
The present inventors obtained, with respect to E. coli K-12, a mutant, thrR (herein referred to as rhtA23) that has resistance to high concentrations of threonine or homoserine in minimal media (Astaurova, O. B. et al., Appl. Bioch. And Microbiol., 21, 611–616 (1985)). The mutation resulted in improvement in production of L-threonine (SU Pat. No. 974817), homoserine, and glutamate (Astaurova, O. B. et al., Appl. Bioch. And Microbiol., 27, 556–561, 1991, EP 1013765 A) by the respective E. coli producing strain, such as the strain VKPM B-3996. Furthermore, the present inventors have revealed that the rhtA gene exists at 18 min on E. coli chromosome close to the glnHPQ operon that encodes components of the glutamine transport system, and that the rhtA gene is identical to ORF1 (ybiF gene, numbers 764 to 1651 in the GenBank accession number AAA218541, gi:440181), located between the pexB and ompX genes. The unit expressing a protein encoded by the ORF1 has been designated as rhtA (rht: resistance to homoserine and threonine) gene. Also, the present inventors have found that the rhtA23 mutation is an A-for-G substitution at position −1 with respect to the ATG start codon (ABSTRACTS of 17th International Congress of Biochemistry and Molecular Biology in conjugation with 1997 Annual Meeting of the American Society for Biochemistry and Molecular Biology, San Francisco, Calif. Aug. 24–29, 1997, abstract No. 457, EP 1013765 A).
Under conditions of optimization of the mainstream threonine biosynthetic pathway, further improvement of threonine-producing strains can be accomplished by supplementing the bacterium with increasing amounts of distant precursors of threonine, such as aspartate.
It is known that aspartate is a carbon donor during synthesis of the amino acids of the aspartate family (threonine, methionine, lysine), and diaminopimelate, a compound constituent of the bacterial cell wall. These syntheses are performed by a complex pathway having several branch points and an extremely sensitive regulatory scheme. In the branch points (aspartate, aspartate semialdehyde, homoserine), there are as many isozymes as there are amino acids deriving from this biosynthetic step. The aspartokinase homoserine dehydrogenase I encoded by part of thrABC operon causes the first and third reactions of threonine biosynthesis. Threonine and isoleucine regulate the expression of aspartokinase homoserine dehydrogenase I, and threonine inhibits both activities to catalyze the above-described reactions (Escherichia coli and Salmonella, Second Edition, Editor in Chief: F. C. Neidhardt, ASM Press, Washington D.C., 1996).
The asd gene encodes aspartate-β-semialdehyde dehydrogenase (Asd; EC 1.2.1.11), which is a key enzyme in the biosynthetic pathways for lysine, methionine, threonine and diaminopimelate. Aspartate-β-semialdehyde dehydrogenase reversibly converts L-aspartyl-4-P to L-aspartate semialdehyde along with the reduction of NADP. The effect of amplification of the asd gene on production of L-lysine, an amino acid of aspartate family, by E. coli strain is disclosed (U.S. Pat. No. 6,040,160). It has also been disclosed that aspartate-β-semialdehyde dehydrogenase could be useful for production of L-lysine, L-threonine and L-isoleucine by coryneform bacteria (European patent application 0219027).
However, there has been no report to date of using a bacterium belonging to the genus Escherichia with enhanced aspartate-β-semialdehyde dehydrogenase activity for the production of L-threonine.